Identifying Antigen-Specific T Cells in ANCA-Associated Vasculitis: A Glimpse of the Future?
Authors: Lani Shochet, A. Richard Kitching
Journal: Journal of the American Society of Nephrology (2022)

ANCA-associated vasculitis (AAV) is an important cause of rapidly progressive autoimmune GN.1 The hallmark clinical characteristic of AAV is serum ANCA specific for either proteinase 3 (PR3) or myeloperoxidase. ANCA induces neutrophil activation and recruitment to vulnerable vascular beds, particularly the glomerulus, where neutrophils induce injury. Although B cell autoreactivity assessed by ANCA is a key clinical diagnostic test in AAV, CD4+ T cells are also intricately involved (Figure 1). As PR3 and myeloperoxidase are T cell–dependent antigens, antigen-specific T follicular helper cells promote ANCA production by B cells. In addition, effector CD4+ T cells mediate tissue damage, with injury being induced by cytotoxic T cells and antigen-specific T helper 1 and T helper 17 cells. Lastly, CD4+ regulatory T cells are likely to be important in immunologic tolerance, as in other forms of vasculitis.2Figure 1.: PR3-AAV and antigen-specific T cells. Currently, decisions on treatment duration and determination of relapse in this autoimmune disease are often difficult and are guided by clinical features, assessment of B cell autoimmunity by ANCA, and other investigations (left). The ability to define active autoimmunity more precisely by identifying and phenotyping PR3-specific CD4+ T cells (right) would allow for a more thorough understanding of disease course, paving the way for more precise treatment options, by examining the numbers, phenotyping, and activity of autoreactive T cells. Tfh, T follicular helper cell; Th, T helper cell; Treg, regulatory CD4+ T cell. Figure created with BioRender.com.Why is studying CD4+ T cells in AAV important? Given their roles in AAV, CD4+ T cell research in AAV is relevant in understanding the biology of disease, in developing better ways of monitoring disease and in moving toward antigen-specific therapies. The re-emergence of measurable B cell autoimmunity, in the form of PR3-ANCA, increases the risk of relapse, but ANCA cannot be used alone to dictate therapy. Assessing both T and B cell autoimmunity with other markers of inflammation, may allow for better definition of immunologic remission and improved assessment of the risk and detection of relapse.3 In this issue of JASN Chen et al.3 begin to address this issue by examining PR3-specific CD4+ T cells in PR3-AAV. As in other autoimmune diseases, the presence of particular MHC class II (MHCII) allomorphs confers an increased risk of developing AAV, particularly in PR3-AAV.4 HLA-DPB1*04:01 (typically associated with HLA-DPA1*01 as "HLA-DP4") is over-represented in people with PR3-AAV and also confers an increased risk of disease relapse.5 Analyses of risk HLA allomorphs suggest that amino acid commonalities in specific peptide binding positions define the HLA-associated risk of disease.6 In some populations, HLA-DR15 is also associated with disease.6,7 How does a particular HLA modulate the risk of developing disease or the rates of relapse? There are several potential mechanisms (reviewed by Dendrou et al.8), but perhaps the most well-known mechanism is the binding of specific peptides to the relevant HLA that permits CD4+ T cells to escape negative selection in the thymus, allowing autoreactive T cells to exist in secondary lymphoid organs. Subsequently, under some conditions, these cells can be activated by antigen presenting cells expressing cognate peptide bound to the relevant HLA. Thus, not only is the binding of a particular peptide important, but so is the relationship between the peptide-HLA complex and T cell receptor. Subtle differences in the peptide-MHCII complex determined by the HLA allomorph, antigenic peptide, or both are detected by the T cell receptor, thereby influencing selection, clonal expansion, T cell repertoire. and T cell subset polarity, cytotoxicity, and memory. After confirming an increased relapse rate in the presence of HLA-DP45 in their patient cohort, Chen et al.3 selected a PR3 peptide (PR3225–239) with high affinity for DP4 and identified PR3225–239 specific CD4+ T cells in patients with PR3-AAV both on treatment and in long-term remission off treatment. The identification of antigen-specific CD4+ T cells can be difficult, as despite their central role in immunity, the frequency of antigen-specific cells is low. CD4+ T cells specific for a particular peptide can be identified by flow cytometry using HLA class II-peptide multimers (tetramers). Tetramer-stained PR3225–239-specific CD4+ T cells were present in approximately 50% of PR3-AAV patients after culture with PR3225–239, with the need for ex vivo expansion suggesting that PR3225–239-specific CD4+ T cells may be present only at very low frequencies in vivo. In two of four patients frequencies of peptide-specific T cells fell in remission, whereas cells from two of four patients on treatment made IFN-γ after stimulation with PR3225–239. The authors proposed that the interactions of PR3225–239 with HLA-DP4 differ in people with PR3-AAV on treatment from those in patients in long-term remission off therapy. They incubated cells from HLA-DP4+ patients with fluorescently labeled PR3225–239. In those on therapy, over 40% of DPB1 cells also stained positive for PR3225–239, whereas this figure was only 10%–15% in healthy control subjects or patients in long-term remission off therapy. Although dual staining of HLA-DP+ cells incubated with fluorescent PR3225–239 suggests that the signal from these cells reflects HLA-DP4–bound PR3225–239, colocalization could be definitively demonstrated by quantifying HLA-peptide complexes using specialized mass spectrometry techniques to identify HLA-DP4–bound peptides.9 Why did people on treatment exhibit enhanced PR3225–239 fluorescence? Although it may represent a shift in PR3225–239 presentation by DP4 according to disease stage, there are other possible explanations. People on treatment may have been B cell deplete, altering the populations of HLA-DP–expressing cells and thus, differentially affecting the capacity of fluorescenated PR3225–239 to bind to the cells' surface. Immunosuppressive therapy might have influenced the findings in other ways: for example, by altering the strength of binding of endogenous peptides to HLA, making them more easily displaced by exogenous high-affinity PR3225–239. Lastly, results obtained by pulsing cells with an exogenous peptide may or may not be applicable to in vivo biology, where proteins are engulfed by antigen presenting cells, intracellularly processed into short peptides, and then, loaded into MHCII and MHCII-peptide complexes transported to the cell surface. Chen et al.3 used tetramers to identify autoreactive PR3225–239-specific CD4+ T cells, but is PR3225–239 a key epitope or even the immunodominant PR3 CD4+ T cell epitope? Tetramer staining cannot address this question, and not all patients with PR3-AAV had proportions of tetramer-positive cells above baseline. The selection of PR3225–239 for testing, while reasonable, was based on peptide/HLA-DP4 binding, leading one to infer that the strongest binding PR3 peptide represents the key PR3 epitope. This is possible, although not always the case, as in vivo antigen processing and the affinity/avidity of the peptide-HLA binding to the T cell receptor influence the selection and activation of CD4+ T cells. Furthermore, as antigen presentation involves processing of extracellular proteins, intracellular processing influences the library of PR3 peptides generated for loading into DP4. Studies using overlapping peptide libraries have been informative in other forms of renal vasculitis and may help determine immunodominant epitopes in PR3-AAV. Progress is needed in AAV so that patients can be effectively treated with the most appropriate therapy for the optimal length of time. Unlike detecting autoantibodies, identifying antigen-specific T cells is challenging, and techniques would need to be refined for T cell assessment to become part of clinical practice. Nonetheless, these studies are an initial step in understanding the relationship between antigenic PR3 peptides, HLA, and autoreactive CD4+ T cells. Chen et al.3 provide a glimpse into the potential for identification of antigen-specific T cells to not only improve our understanding of PR3-AAV but also, signpost the way to more precise therapy. Disclosures A.R. Kitching reports consultancy agreements with Toleranzia (consulting payments to institution), Variant Bio (consulting payments to institution), and Visterra Inc. (consulting payments to self and to institution); research funding from CSL Limited, Vifor Pharma, and Visterra Inc.; honoraria from Vifor Pharma (seminar); advisory or leadership role as cochair of the Australia and New Zealand Society of Nephrology Research Advisory Committee (to November 2021) and chair of the Australian and New Zealand Vasculitis Society; serving on the editorial boards CJASN, JASN, Internal and Emergency Medicine, and Kidney International; serving as an associate editor (Autoimmunity Section) for Frontiers in Immunology and a section editor for Nephrology (to the end of 2021); and other interests or relationships as a member of the Kidney Health Australia Research Advisory Group. The remaining author has nothing to disclose Funding A.R. Kitching is funded by National Health and Medical Research Council investigator grant 2008921. L. Shochet is supported by Australian National Health and Medical Research Council Postgraduate Research Scholarship grant 1151380 and the Royal Australasian College of Physicians Jacquot Award.